Factor VII glycoforms

ABSTRACT

The present invention provides preparations of Factor VIIa polypeptides or Factor VIIa-related polypeptides that exhibit predetermined glycoform patterns. The preparations of the invention exhibit improved functional properties and are useful for treating Factor VII-mediated conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/969,357 filed Oct. 2, 2001 which claims priority under 35 U.S.C. 119of Danish applications nos. PA 2000 01456 filed on Oct. 2, 2000; PA 200100262 filed Feb. 16, 2001; PA 2001 00430 filed Mar. 14, 2001; and PA2001 00751 filed on May 14, 2001 and U.S. provisional applications Nos.60/238,944 filed on Oct. 10, 2000; 60/271,581 filed on Feb. 26, 2001;and 60/276,322 filed on Mar. 16, 2001, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions comprising Factor VII andother blood clotting factors having altered patterns ofasparagine-linked glycosylation.

BACKGROUND OF THE INVENTION

The proteins involved in the clotting cascade, including, e.g., FactorVII, Factor VIII, Factor IX, Factor X, and Protein C, are proving to beuseful therapeutic agents to treat a variety of pathological conditions.Accordingly, there is an increasing need for formulations comprisingthese proteins that are pharmaceutically acceptable and exhibit auniform and predetermined clinical efficacy.

Because of the many disadvantages of using human plasma as a source ofpharmaceutical products, it is preferred to produce these proteins inrecombinant systems. The clotting proteins, however, are subject to avariety of co- and post-translational modifications, including, e.g.,asparagine-linked (N-linked) glycosylation; O-linked glycosylation; andγ-carboxylation of glu residues. These modifications may bequalitatively or quantitatively different when heterologous cells areused as hosts for large-scale production of the proteins. In particular,production in heterologous cells often results in a different array ofglycoforms, which are identical polypeptides having different covalentlylinked oligosaccharide structures.

In different systems, variations in the oligosaccharide structure oftherapeutic proteins have been linked to, inter alia, changes inimmunogenicity and in vivo clearance. Thus, there is a need in the artfor compositions and methods that provide clotting protein preparations,particularly preparations comprising recombinant human Factor VII,modified Factor VII, or Factor VII-related polypeptides, that containpredetermined glycoform patterns.

SUMMARY OF THE INVENTION

The present invention relates to preparations comprising Factor VIIpolypeptides or Factor VII-related polypeptides that exhibitpredetermined glycoform patterns. As used herein, a Factor VII or FactorVII-related preparation refers to a plurality of Factor VII or FactorVII-related polypeptides, including variants and chemically modifiedforms, as well as forms that have been proteolytically activated (e.g.,Factor VIIa), that have been separated from the cell in which they weresynthesized. A glycoform pattern refers to the distribution within thepreparation of oligosaccharide chains having varying structures that arecovalently linked to Factor VII polypeptides or Factor VII-relatedpolypeptides.

In one aspect, the invention provides a preparation comprising aplurality of Factor VII polypeptides or Factor VII-related polypeptides,wherein the polypeptides comprise asparagine-linked oligosaccharidechains and wherein one or more of the following applies: (i) betweenabout 94-100% of the oligosaccharide chains comprise at least one sialicacid moiety; (ii) between about 0-7% of the oligosaccharide chains havea neutral charge; (iii) less than about 16%, such as, e.g., betweenabout 6-16% of the oligosaccharide chains comprise at least one terminalgalactose residue; (iv) less than about 25%, such as, e.g., betweenabout 6-9% of the oligosaccharide chains comprise at least one terminalN-acetylgalactosamine residue; or (v) less than about 30%, such as,e.g., between about 11-23% of the oligosaccharide chains comprise atleast one terminal galactose or N-acetylgalactosamine residue. In someembodiments, in addition to one or more of (i)-(v): all of the sialicacid residues in the oligosaccharide chains are linked to galactose viaan α2->3 linkage; at least some of the sialic acid residues compriseN-glycolylneuraminic acid (Neu5Gc) in addition to N-acetylneuraminicacid (Neu5Ac); and/or the oligosaccharide chains comprise fucoseresidues linked α1->6 to a core N-acetylglucosamine. In one embodiment,the invention encompasses a preparation comprising wild-type Factor VIIain which between about 94-100% of the oligosaccharide chains have atleast one sialic acid residue and all of the sialic acid residues arelinked to galactose via an α2->3 linkage. In another embodiment, theinvention encompasses a preparation comprising wild-type Factor VIIa inwhich between about 94-100% of the oligosaccharide chains have at leastone sialic acid residue and at least some of the sialic acid residuesare N-glycolylneuraminic acid. In yet another embodiment, the inventionencompasses a preparation comprising wild-type Factor VIIa in whichbetween about 94-100% of the oligosaccharide chains have at least onesialic acid residue and at least some of the chains containN-acetylgalactosamine. The preparations of the present invention thus donot encompass wild-type Factor VII or Factor VIIa that has been isolatedfrom human plasma and has not been modified ex vivo by glycosidasetreatment.

In another aspect, the invention provides a preparation comprising aplurality of Factor VII polypeptides or Factor VII-related polypeptides,wherein the polypeptides comprise asparagines-linked oligosaccharidechains and wherein at least about 2% of the oligosaccharide chainscontain at least one fucose linked α1->3 to an antennaryN-acetylglucosamine residue (i.e., an N-acetylglucosamine residue thatis linked β1->2,4, or 6 to a Man residue). Preferably, at least about 5%of the oligosaccharide chains contain at least one such antennary fucoseresidue; more preferably, at least about 10% or 20%; and mostpreferably, at least about 40%.

The preparations according to invention may comprise one or more ofunmodified wild-type Factor VII; wild-type Factor VII that has beensubjected to chemical and/or enzymatic modification; and Factor VIIvariants having one or more alterations in amino acid sequence relativeto wild-type Factor VII. The preparations of the invention may bederived from human cells expressing Factor VII from an endogenous FactorVII gene or from cells programmed to express Factor VII or a FactorVII-related polypeptide from a recombinant gene.

In another aspect, the invention provides preparations comprising FactorVII or Factor VII-related polypeptides that exhibit one or more improvedfunctional properties, including, without limitation, increased storagestability, bioavailability, half-life, and/or tissue factor-independentthrombin generating activity. In one embodiment, a Factor VIIpreparation comprising asparagine-linked oligosaccharide chains in whichat least about 2% of the oligosaccharide chains contain at least onefucose linked α1->3 to an antennary N-acetylglucosamine residue exhibitstissue factor-independent thrombin generating activity that is at leastabout 110% that of a reference preparation, preferably at least about125% and most preferably at least about 140%, when the oligosaccharidesof the reference preparation lack fucose linked α1->3 to an antennaryN-acetylglucosamine.

In another aspect, the invention encompasses methods for determiningand/or optimizing the glycoform pattern of Factor VII and FactorVII-related polypeptides, which are carried out by the steps of:

(a) culturing a cell expressing Factor VII or Factor VII-relatedpolypeptides under a first set of predetermined culture conditions;

(b) recovering Factor VII or Factor VII-related polypeptides from theculture to obtain a preparation comprising the polypeptides; and

(c) analyzing the structure of the oligosaccharides linked to thepolypeptides to determine the glycoform pattern of the preparation.

The methods may further comprise altering the culture conditions of step(a) to achieve a second set of predetermined culture conditions; andrepeating the steps until a desired glycoform pattern is achieved.Alternatively, the methods may further comprise treating the preparationchemically or enzymatically to alter the oligosaccharide structure; andrepeating the steps until a desired glycoform pattern is achieved.Furthermore, the methods may comprise the additional steps of subjectingpreparations having predetermined glycoform patterns to at least onetest of bioactivity or other functionality (such as, e.g.,pharmacokinetic profile or stability), and correlating particularglycoform patterns with particular bioactivity or functionalityprofiles.

In another aspect, the invention provides methods for producing apreparation comprising Factor VII polypeptides or Factor VII-relatedpolypeptides having a predetermined pattern of N-linked glycosylation.In some embodiments, the methods are carried out by culturing a cellexpressing the polypeptides under conditions in which at least about 94%of the asparagine-linked oligosaccharides linked to the Factor VIIpolypeptides or Factor VII-related polypeptides comprise at least onesialic acid residue, e.g., one, two, three, or four sialic acidresidues. In some embodiments, the methods are carried out by culturinga cell expressing the polypeptides under conditions in which at leastabout 5% of the oligosaccharide chains contain at least one fucoselinked α1->3 to an antennary N-acetylglucosamine residue. In someembodiments, Factor VII polypeptides or Factor VII-related polypeptides,irrespective of their source, are subjected to enzymatic treatments toachieve the desired glycoform patterns.

In another aspect, the invention provides pharmaceutical formulationscomprising the preparations of the invention and methods of preventingand/or treating syndromes that are responsive to Factor VII polypeptidesor Factor VII-related polypeptides. The methods comprise administeringthe pharmaceutical formulations to a patient in need of treatment, underconditions that result in either an enhancement or inhibition in bloodclotting. In one series of embodiments, Factor VII preparations areadministered when it is desired to enhance blood clotting, such as,e.g., in haemophilia A, haemophilia B, Factor XI deficiency, Factor VIIdeficiency, thrombocytopenia, or von Willebrand's disease; in syndromesaccompanied by the presence of a clotting factor inhibitor; before,during, or after surgery or anticoagulant therapy; or after trauma. Inanother series of embodiments, preparations of Factor VII-relatedpolypeptides (i.e., preparations having reduced or modified bioactivityrelative to wild-type Factor VII) are administered to reduce bloodclotting, such as, e.g., in patients undergoing angioplasty or thosesuffering from deep vein thrombosis, pulmonary embolism, stroke,disseminated intravascular coagulation (DIC), fibrin deposition in lungsand kidneys associated with gram-negative endotoxemia, or myocardialinfarction. According to the invention, preparations of FactorVII-related polypeptides may also be administered when it is desired tomodify, such as, e.g., reduce, intracellular signalling via a tissuefactor (TF)-mediated pathway, to treat conditions such as, e.g., AcuteRespiratory Distress Syndrome (ARDS), Systemic Inflammatory ResponseSyndrome (SIRS), Hemolytic Uremic Syndrome (HUS), Multiple Organ Failure(MOF), and thrombocytopenia purpura (TTP).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that preparations of coagulationproteins having predetermined glycoform patterns exhibit improvedfunctional properties. Accordingly, the present invention relates tomethods and compositions that provide these protein preparations. Inparticular, the invention relates to preparations comprising Factor VIIpolypeptides and Factor VII-related polypeptides having specificpredetermined patterns of asparagine-linked (N-linked) oligosaccharides.The preparations of the invention exhibit altered properties, including,without limitation, improved pharmacokinetic properties and improvedclinical efficacy. The invention also encompasses pharmaceuticalformulations that comprise these preparations, as well as therapeuticmethods that utilize the formulations.

Factor VII Polypeptides and Factor VII-Related Polypeptides

The present invention encompasses human Factor VII polypeptides, suchas, e.g., those having the amino acid sequence disclosed in U.S. Pat.No. 4,784,950 (wild-type Factor VII). As used herein, “Factor VII” or“Factor VII polypeptide” encompasses wild-type Factor VII, as well asvariants of Factor VII exhibiting substantially the same or improvedbiological activity relative to wild-type Factor VII. The term “FactorVII” is intended to encompass Factor VII polypeptides in their uncleaved(zymogen) form, as well as those that have been proteolyticallyprocessed to yield their respective bioactive forms, which may bedesignated Factor VIIa. Typically, Factor VII is cleaved betweenresidues 152 and 153 to yield Factor VIIa.

As used herein, “Factor VII-related polypeptides” encompassespolypeptides, including variants, in which the Factor VIIa biologicalactivity has been substantially modified or reduced relative to theactivity of wild-type Factor VIla. These polypeptides include, withoutlimitation, Factor VII or Factor VIIa that has been chemically modifiedand Factor VII variants into which specific amino acid sequencealterations have been introduced that modify or disrupt the bioactivityof the polypeptide.

The biological activity of Factor VIIa in blood clotting derives fromits ability to (i) bind to tissue factor (TF) and (ii) catalyze theproteolytic cleavage of Factor IX or Factor X to produce activatedFactor IX or X (Factor IXa or Xa, respectively). For purposes of theinvention, Factor VIIa biological activity may be quantified bymeasuring the ability of a preparation to promote blood clotting usingFactor VII-deficient plasma and thromboplastin, as described, e.g., inU.S. Pat. No. 5,997,864. In this assay, biological activity is expressedas the reduction in clotting time relative to a control sample and isconverted to “Factor VII units” by comparison with a pooled human serumstandard containing 1 unit/ml Factor VII activity. Alternatively, FactorVIIa biological activity may be quantified by (i) measuring the abilityof Factor VIIa to produce of Factor Xa in a system comprising TFembedded in a lipid membrane and Factor X. (Persson et al., J. Biol.Chem. 272:19919-19924, 1997); (ii) measuring Factor X hydrolysis in anaqueous system (see, Example 5 below); (iii) measuring its physicalbinding to TF using an instrument based on surface plasmon resonance(Persson, FEBS Letts. 413:359-363, 1997) (iv) measuring hydrolysis of asynthetic substrate (see, Example 4 below); and (v) measuring generationof thrombin in a TF-independent in vitro system.

Factor VII variants having substantially the same or improved biologicalactivity relative to wild-type Factor VIIa encompass those that exhibitat least about 25%, preferably at least about 50%, more preferably atleast about 75% and most preferably at least about 90% of the specificactivity of wild-type Factor VIIa that has been produced in the samecell type, when tested in one or more of a clotting assay, proteolysisassay, or TF binding assay as described above. Factor VII variantshaving substantially reduced biological activity relative to wild-typeFactor VIIa are those that exhibit less than about 25%, preferably lessthan about 10%, more preferably less than about 5% and most preferablyless than about 1% of the specific activity of wild-type Factor VIIathat has been produced in the same cell type when tested in one or moreof a clotting assay, proteolysis assay, or TF binding assay as describedabove. Factor VII variants having a substantially modified biologicalactivity relative to wild-type Factor VII include, without limitation,Factor VII variants that exhibit TF-independent Factor X proteolyticactivity and those that bind TF but do not cleave Factor X.

Variants of Factor VII, whether exhibiting substantially the same orbetter bioactivity than wild-type Factor VII, or, alternatively,exhibiting substantially modified or reduced bioactivity relative towild-type Factor VII, include, without limitation, polypeptides havingan amino acid sequence that differs from the sequence of wild-typeFactor VII by insertion, deletion, or substitution of one or more aminoacids. Non-limiting examples of Factor VII variants having substantiallythe same biological activity as wild-type Factor VII include S52A-FVIIa,S60A-FVIIa (lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998);FVIIa variants exhibiting increased proteolytic stability as disclosedin U.S. Pat. No. 5,580,560; Factor VIIa that has been proteolyticallycleaved between residues 290 and 291 or between residues 315 and 316(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); and oxidizedforms of Factor VIIa (Komfelt et al., Arch. Biochem. Biophys. 363:43-54,1999). Non-limiting examples of Factor VII variants having substantiallyreduced or modified biological activity relative to wild-type Factor VIIinclude R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990),S344A-FVIIa (Kazama et al., J. Biol. Chem. 270:66-72, 1995), FFR-FVIIa(Hoist et al., Eur. J Vasc. Endovasc. Surg. 15:515-520, 1998), andFactor VIIa lacking the Gla domain, (Nicolaisen et al., FEBS Letts.317:245-249, 1993). Non-limiting examples of chemically modified FactorVII polypeptides and sequence variants are described, e.g., in U.S. Pat.No. 5,997,864.

Asparagine-Linked Glycosylation

The present invention provides preparations of Factor VII polypeptidesor Factor VII-related polypeptides that comprise a particular spectrumof Factor VII glycoforms, i.e., Factor VII polypeptides or FactorVII-related polypeptides having predetermined patterns ofasparagine-linked (N-linked) oligosaccharide chains.

As used herein, a “pattern” of N-linked glycosylation or a glycoform“pattern”, “distribution”, or “spectrum” refers to the representation ofparticular oligosaccharide structures within a given population ofFactor VII polypeptides or Factor VII-related polypeptides. Non-limitingexamples of such patterns include the relative proportion ofoligosaccharide chains that (i) have at least one sialic acid residue;(ii) lack any sialic acid residues (i.e., are neutral in charge); (iii)have at least one terminal galactose residue; (iv) have at least oneterminal N-acetylgalactosamine residue; (v) have at least one “uncapped”antenna, i.e., have at least one terminal galactose orN-acetylgalactosamine residue; or (vi) have at least one fucose linkedα1->3 to an antennary N-acetylglucosamine residue.

As used herein, an oligosaccharide chain refers to the entireoligosaccharide structure that is covalently linked to a singleasparagine residue. Factor VII is normally glycosylated at Asn 145 andAsn 322. An N-linked oligosaccharide chain present on Factor VIIproduced in a human in situ may be bi-, tri, or tetraantennary, witheach antenna having the structure Neu5Ac(α2->3 or α2->6)Gal(β1->4)GlcNAc linked (β1->2,4, or 6) to a Man residue which is linked (α1->3 or6) to Man(β->4)GlcNAc(β1->4)GlcNAc-Asn. (Neu5Ac signifiesN-acetylneuraminic acid (sialic acid), Gal signifies galactose, GlcNAcsignifies N-acetylglucosamine, and Man signifies mannose). Theoligosaccharide chains may also comprise fucose residues, which may belinked α1->6 to GlcNAc. When Factor VII is produced in a human in situ,some of the oligosaccharide chains lack core fucose residues; all of thechains lack antennary fucose residues; and all of the chains are almostcompletely sialylated, i.e., the terminal sugar of each antenna isN-acetylneuraminic acid linked to galactose via an α2->3 or α2->6linkage.

When produced in other circumstances, however, Factor VII may containoligosaccharide chains having different terminal structures on one ormore of their antennae, such as, e.g., lacking sialic acid residues;containing N-glycolylneuraminic acid (Neu5Gc) residues; containing aterminal N-acetylgalactosamine (GalNAc) residue in place of galactose;and the like. When produced in, e.g., BHK cells cultured in the presenceof calf serum, Factor VII preparations exhibit the followingoligosaccharide patterns:

-   -   87-93% of the oligosaccharide chains contain at least a single        sialic acid residue;    -   7-13% are neutral (lack any sialic acid);    -   9-16% contain at least one terminal galactose residue;    -   19-29% contain at least one terminal N-acetylgalactosamine        residue; and    -   30-39% contain at least one uncapped antenna, i.e., contain at        least one terminal galactose or N-acetylgalactosamine residue.

The present inventors have produced Factor VII preparations containingspecific predetermined oligosaccharide patterns that differ from thosepreviously described. The present invention encompasses preparationscomprising Factor VII polypeptides or Factor VII-related polypeptidesexhibiting one or more of the following glycoform patterns:

(i) Between about 94-100% of the oligosaccharide chains contain at leastone sialic acid residue, such as, e.g., between about 94-99%, betweenabout 95-98%, or between about 96-97%. In different embodiments, atleast about 94%, 95%, 96%, or 97% of the oligosaccharide chains containat least one sialic acid residue.

(ii) 6% or less of the oligosaccharide chains are neutral, such as,e.g., between about 1.5-6% or between about 2-4%.

(iii) Less than about 16%, preferably, less than about 10% of theoligosaccharide chains contain at least one terminal galactose, such as,e.g., between about 6-10% or between about 8-9%;

(iv) Less than about 25%, preferably, less than about 10% of theoligosaccharide chains contain at least one terminal GalNAc residue,such as, e.g., between about 6-9% or between about 7-8%;

(v) Less than about 30, preferably, less than about 25% of theoligosaccharide chains contain at least one uncapped antenna, such as,e.g., between about 11-23% or between about 12-18%; and

(vi) At least about 2%, preferably, at least about 5%, more preferably,at least about 10% or 20%; and most preferably, at least about 40%, ofthe oligosaccharide chains contain at least one fucose linked α1->3 toan antennary N-acetylglucosamine residue (i.e., an N-acetylglucosamineresidue that is linked β1->2,4, or 6 to a Man residue).

It will be understood that each of (i)-(vi) may represent a distinctglycoform pattern that is encompassed by the present invention, i.e., apreparation according to the invention may be described by only one of(i)-(vi). Alternatively, depending on the particular glycoform pattern,a preparation encompassed by the invention may be described by more thanone of (i)-(vi).

Furthermore, a preparation encompassed by the invention may be describedby one or more of (i)-(vi) in combination with one or more otherstructural features. For example, the invention encompasses preparationscomprising Factor VII polypeptides or Factor VII-related polypeptides inwhich the sialic acid residues (Neu5Ac or Neu5Gc) are linked togalactose exclusively in an α2->3 configuration. The invention alsoencompasses preparations comprising Factor VII polypeptides or FactorVII-related polypeptides that contain fucose linked a 1->6 to a coreN-acetylglucosamine and/or fucose linked α1->3 to an antennaryN-acetylglucosamine. In one series of embodiments, the preparations ofthe invention encompass Factor VII or Factor VII-related polypeptides inwhich more than 99% of the oligosaccharide chains contain at least onesialic acid residue and (a) the sialic acid residues are linkedexclusively in an α2->3 configuration and/or (b) there are fucoseresidues linked to core N-acetylglucosamines and/or (c) a detectablenumber of antenna terminate in N-acetylgalactosamine. In one embodiment,the invention encompasses preparations comprising wild-type Factor VIIain which more than 99% of the oligosaccharide chains contain at leastone sialic acid residue and the sialic acid residues are linked togalactose exclusively in an α2->3 configuration. In another embodiment,the invention encompasses preparations comprising wild-type Factor VIIain which more than 99% of the oligosaccharide chains contain at leastone sialic acid residue and at least some of the oligosaccharide chainscomprise N-acetylgalactosamine. The present invention does not encompasswild-type Factor VII or wild-type Factor VIIa that is isolated fromhuman plasma and is not modified ex vivo by treatment with glycosidases.

In one embodiment, the Factor VIIa preparation comprises oligosaccharidechains having a single fucose linked α1->3 to one antennaryN-acetylglucosamine. In another embodiment, the Factor VIIa preparationcomprises oligosaccharide chains having fucose residues linked α1->3 toeach antennary N-acetylglucosamine of a biantennary oligosaccharide(Sialyl Lewis X structure). In another embodiment, the Factor VIlapreparation comprises oligosaccharide chains having (i) a fucose linkedto a core N-acetylglucosamine and (ii) a single fucose linked α1->3 toone antennary N-acetylglucosamine. In another embodiment, the FactorVIIa preparation comprises oligosaccharide chains having (i) a fucoselinked to a core N-acetylglucosamine and (ii) fucose residues linkedα1->3 to each antennary N-acetylglucosamine of a biantennaryoligosaccharide.

In practicing the present invention, the pattern of N-linkedoligosaccharides may be determined using any method known in the art,including, without limitation: high-performance liquid chromatography(HPLC); capillary electrophoresis (CE); nuclear magnetic resonance(NMR); mass spectrometry (MS) using ionization techniques such asfast-atom bombardment, electrospray, or matrix-assisted laser desorption(MALDI); gas chromatography (GC); and treatment with exoglycosidases inconjunction with anion-exchange (AIE)-HPLC, size-exclusionchromatography (SEC), or MS. See, e.g., Weber et al., Anal. Biochem.225:135 (1995); Klausen et al., J. Chromatog. 718:195 (1995); Morris etal., in Mass Spectrometry of Biological Materials, McEwen et al., eds.,Marcel Dekker, (1990), pp 137-167; Conboy et al., Biol. Mass Spectrom.21:397, 1992; Hellerqvist, Meth. Enzymol. 193:554 (1990); Sutton et al.,Anal. Biohcem. 318:34 (1994); Harvey et al., Organic Mass Spectrometry29:752 (1994).

Following resolution of Factor VII-derived oligosaccharide chains usingany of the above methods (or any other method that resolvesoligosaccharide chains having different structures), the resolvedspecies are assigned, e.g., to one of groups (i)-(v). The relativecontent of each of (i)-(v) is calculated as the sum of theoligosaccharides assigned to that group relative to the total content ofoligosaccharide chains in the sample.

For example, using AIE-HPLC, 13 or more N-linked oligosaccharide peakscan be resolved from a recombinant Factor VII preparation produced inBHK cells. See, e.g., Klausen et al., Mol. Biotechnol. 9:195, 1998. Fiveof the peaks (designated 1-5 in Klausen et al.) do not contain sialicacid, while eight of the peaks (designated 6, 7, and 10-15) do containsialic acid.

It will be understood that, in a given analysis, the number anddistribution of sialic acid-containing and sialic acid-lacking chainsmay depend upon (a) the polypeptide being expressed; (b) the cell typeand culture conditions; and (c) the method of analysis that is employed,and that the resulting patterns may vary accordingly.

In any case, once the sialic acid-containing oligosaccharides have beenresolved from the non-sialic acid-containing oligosaccharides,conventional data analysis programs are used to calculate the area undereach peak; the total peak area; and the percentage of the total peakarea represented by a particular peak. In this manner, for a givenpreparation, the sum of the areas of sialic acid-containing peaks/totalpeak area ×100 yields the % sialylation value for the preparationaccording to the present invention (i.e., the proportion ofoligosaccharide chains having at least one sialic acid residue). In asimilar manner, the % of chains having no sialic acid or at least onegalactose or N-acetylglucosamine can be calculated.

Methods for Producing Factor VII Preparations Having a PredeterminedPattern of N-linked Oligosaccharides

Preparations of Factor VII, Factor VII variants, or Factor VII-relatedpolypeptides, each having a predetermined pattern of N-linkedoligosaccharides, may be produced using any appropriate host cell thatexpresses Factor VII or Factor VII-related polypeptides.

Host cells: In some embodiments, the host cells are human cellsexpressing an endogenous Factor VII gene. In these cells, the endogenousgene may be intact or may have been modified in situ, or a sequenceoutside the Factor VII gene may have been modified in situ to alter theexpression of the endogenous Factor VII gene. Any human cell capable ofexpressing an endogenous Factor VII gene may be used.

In other embodiments, heterologous host cells are programmed to expresshuman Factor VII from a recombinant gene. The host cells may bevertebrate, insect, or fungal cells. Preferably, the cells are mammaliancells capable of the entire spectrum of mammalian N-linkedglycosylation; O-linked glycosylation; and γ-carboxylation. See, e.g.,U.S. Pat. Nos. 4,784,950. Preferred mammalian cell lines include the CHO(ATCC CCL 61), COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) andHEK293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977)cell lines. A preferred BHK cell line is the tk⁻ ts13 BHK cell line(Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982),hereinafter referred to as BHK 570 cells. The BHK 570 cell line isavailable from the American Type Culture Collection, 12301 Parklawn Dr.,Rockville, Md. 20852, under ATCC accession number CRL 10314. A tk⁻ ts13BHK cell line is also available from the ATCC under accession number CRL1632. In addition, a number of other cell lines may be used, includingRat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCCCRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469(ATCC CCL 9.1) and DUKX cells (CHO cell line) (Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220, 1980). (DUKX cells also referred toas CXB11 cells), and DG44 (CHO cell line) (Cell, 33:405, 1983, andSomatic Cell and Molecular Genetics 12:555, 1986). Also useful are 3T3cells, Namalwa cells, myelomas and fusions of myelomas with other cells.In a particularly preferred embodiment, the host cells are BHK 21 cellsthat have been adapted to grow in the absence of serum and have beenprogrammed to express Factor VII. In some embodiments, the cells may bemutant or recombinant cells that express a qualitatively orquantitatively different spectrum of glycosylation enzymes (such as,e.g., glycosyl transferases and/or glycosidases) than the cell type fromwhich they were derived. The cells may also be programmed to expressother heterologous peptides or proteins, including, e.g., truncatedforms of Factor VII. In one embodiment, the host cells are CHO cellsthat have been programmed to co-express both the Factor VII polypeptideof interest (i.e., Factor VII or a Factor-VII-related polypeptide) andanother heterologous peptide or polypeptide such as, e.g., a modifyingenzyme or a Factor VII fragment.

Methods: The present invention encompasses methods for producing apreparation comprising any of the glycoform patterns described above as(i)-(vi) and, in further embodiments, methods for optimizing theglycoform distribution of Factor VII and Factor VII-relatedpolypeptides. These methods are carried out by the steps of:

(a) culturing a cell expressing Factor VII or Factor VII-relatedpolypeptides under a first set of predetermined culture conditions;

(b) recovering Factor VII or FactorVII-related polypeptides from theculture to obtain a preparation comprising the polypeptides; and

(c) analyzing the structure of the oligosaccharides linked to thepolypeptides to determine a glycoform pattern.

The methods may further comprise:

(d1) altering the culture conditions of step (a) to achieve a second setof predetermined culture conditions;

(e1) repeating steps (b)-(d1) until a desired glycoform pattern isachieved.

Alternatively, the methods may further comprise

(d2) treating the preparation chemically and/or enzymatically to alterthe oligosaccharide structure; and

(e2) repeating steps (b)-(d2) until a desired glycoform pattern isachieved.

These methods may further comprise the step of subjecting preparationshaving predetermined glycoform patterns to at least one test ofbioactivity (including, e.g., clotting, Factor X proteolysis, or TFbinding) or other functionality (such as, e.g., pharmacokinetic profileor stability), and correlating particular glycoform patterns withparticular bioactivity or functionality profiles in order to identify adesired glycoform pattern.

The variables in the culture conditions that may be altered in step (d1)include, without limitation: the cell of origin, such as, e.g., a cellderived from a different species than originally used; or a mutant orrecombinant cell having alterations in one or more glycosyltransferasesor glycosidases or other components of the glycosylation apparatus (see,Grabenhorst et al., Glycoconjugate J. 16:81, 1999; Bragonzi et al.,Biochem. Biophys. Acta 1474:273, 2000; Weikert, Nature Biotechnol.17:1116, 1999); the level of expression of the polypeptide; themetabolic conditions such as, e.g., glucose or glutamine concentration;the absence or presence of serum; the concentration of vitamin K;protein hydrolysates, hormones, trace metals, salts as well as processparameters like temperature, dissolved oxygen level and pH.

The enzymatic treatments that may be used in step (d2) to modify theoligosaccharide pattern of a preparation include, without limitation,treatment with one or more of sialidase (neuraminidase), galactosidase,fucosidase; galactosyl transferase, fucosyl transferase, and/orsialyltransferase, in a sequence and under conditions that achieve adesired modification in the distribution of oligosaccharide chainshaving particular terminal structures. Glycosyl transferases arecommercially available from Calbiochem (La Jolla, Calif.) andglycosidases are commercially available from Glyko, Inc., (Novato,Calif.).

In one series of embodiments, host cells expressing Factor VII or arelated polypeptide are subjected to specific culture conditions inwhich they secrete glycosylated Factor VII polypeptides having thedesired pattern of oligosaccharide structures described above as any of(i)-(vi). Such culture conditions include, without limitation, areduction in, or complete absence of, serum. Preferably, the host cellsare adapted to grow in the absence of serum and are cultured in theabsence of serum both in the growth phase and in the production phase.Such adaptation procedures are described, e.g., in Scharfenberg, et al.,Animal Cell Technology Developments towards the 21^(st) Century, E. C.Beuvery et al. (Eds.), Kluwer Academic Publishers, pp. 619-623, 1995(BHK and CHO cells); Cruz, Biotechnol. Tech. 11:117-120, 1997 (insectcells); Keen, Cytotechnol. 17:203-211, 1995 (myeloma cells); Berg etal., Biotechniques 14:972-978, 1993 (human kidney 293 cells). In apreferred embodiment, the growth medium that is added to the cellscontains no protein or other component that was isolated from an animaltissue or an animal cell culture. See, e.g., Example 1 below. Typically,in addition to conventional components, a medium suitable for producingFactor VII contains Vitamin K at a concentration between 0.1-50mg/liter, which is required for γ-carboxylation of glutamine residues inFactor VII.

In another series of embodiments, the glycoforms of the invention areproduced by subjecting a preparation of Factor VII or Factor VII-relatedpolypeptides to enzymatic and/or chemical modification of the N-linkedoligosaccharides contained therein.

Factor VII Preparations

As used herein, a “Factor VII preparation” refers to a plurality ofFactor VII polypeptides, Factor VIIa polypeptides, or Factor VII-relatedpolypeptides, including variants and chemically modified forms, thathave been separated from the cell in which they were synthesized.

Separation of polypeptides from their cell of origin may be achieved byany method known in the art, including, without limitation, removal ofcell culture medium containing the desired product from an adherent cellculture; centrifugation or filtration to remove non-adherent cells; andthe like.

Optionally, Factor VII polypeptides may be further purified.Purification may be achieved using any method known in the art,including, without limitation, affinity chromatography, such as, e.g.,on an anti-Factor VII antibody column (see, e.g., Wakabayashi et al., J.Biol. Chem. 261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988);hydrophobic interaction chromatography; ion-exchange chromatography;size exclusion chromatography; electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction and the like. See,generally, Scopes, Protein Purification, Springer-Verlag, New York,1982; and Protein Purification, J.-C. Janson and Lars Ryden, editors,VCH Publishers, New York, 1989. Following purification, the preparationpreferably contains less than about 10% by weight, more preferably lessthan about 5% and most preferably less than about 1%, of non-Factor VIIproteins derived from the host cell.

Factor VII and Factor VII-related polypeptides may be activated byproteolytic cleavage, using Factor XIIa or other proteases havingtrypsin-like specificity, such as, e.g., Factor IXa, kallikrein, FactorXa, and thrombin. See, e.g., Osterud et al., Biochem. 11:2853 (1972);Thomas, U.S. Pat. No. 4,456,591; and Hedner et al., J. Clin. Invest.71:1836 (1983). Alternatively, Factor VII may be activated by passing itthrough an ion-exchange chromatography column, such as Mono Q®(Pharmacia) or the like. The resulting activated Factor VII may then beformulated and administered as described below.

Functional Properties of Factor VII Preparations

The preparations of Factor VII polypeptides and Factor VII-relatedpolypeptides having predetermined oligosaccharide patterns according tothe invention exhibit improved functional properties relative toreference preparations. The improved functional properties may include,without limitation, a) physical properties such as, e.g., storagestability; b) pharmacokinetic properties such as, e.g., bioavailabilityand half-life; and c) immunogenicity in humans.

A reference preparation refers to a preparation comprising a polypeptidethat is identical to that contained in the preparation of the inventionto which it is being compared (such as, e.g., wild-type Factor VII or aparticular variant or chemically modified form) except for exhibiting adifferent pattern of asparagine-linked glycosylation. For example,reference preparations typically comprise one or more of the followingglycoform patterns: (i) less than about 93% (such as, e.g. less thanabout 92% or less than about 90%) of the oligosaccharide chains containat least one sialic acid residue; (ii) at least about 6% (such as, e.g.,at least about 7.5% or at least about 10%) of the oligosaccharide chainslack any sialic acid (i.e., are neutral); (iii) at least about 10% (suchas, e.g., at least about 12.5% or at least about 15%) of theoligosaccharide chains contain at least one terminal galactose residue;(iv) at least about 15% (such as, e.g., at least about 20% or at leastabout 25%) of the oligosaccharide chains contain at least one terminalN-acetylgalactosamine residue; (v) at least about 25% (such as, e.g., atleast about 30% or at least about 35%) of the oligosaccharide chainscontain at least one uncapped antenna (i.e., contain at least oneterminal galactose or N-acetylgalactosamine residue); or (vi)essentially undetectable levels (such as, e.g., less than about 0.2%) ofantennary fucose residues.

Storage stability of a Factor VII preparation may be assessed bymeasuring (a) the time required for 20% of the bioactivity of apreparation to decay when stored as a dry powder at 25° C. and/or (b)the time required for a doubling in the proportion of Factor VIIaaggregates in the preparation.

In some embodiments, the preparations of the invention exhibit anincrease of at least about 30%, preferably at least about 60% and morepreferably at least about 100%, in the time required for 20% of thebioactivity to decay relative to the time required for the samephenomenon in a reference preparation, when both preparations are storedas dry powders at 25° C. Bioactivity measurements may be performed usingany of a clotting assay, proteolysis assay, TF-binding assay, orTF-independent thrombin generation assay.

In some embodiments, the preparations of the invention exhibit anincrease of at least about 30%, preferably at least about 60%, and morepreferably at least about 100%, in the time required for doubling ofaggregates relative to a reference preparation, when both preparationsare stored as dry powders at 25° C. The content of aggregates isdetermined by gel permeation HPLC on a Protein Pak 300 SW column(7.5×300 mm) (Waters, 80013) as follows. The column is equilibrated withEluent A (0.2 M ammonium sulfate, 5 % isopropanol, pH adjusted to 2.5with phosphoric acid, and thereafter pH is adjusted to 7.0 withtriethylamine), after which 25 μg of sample is applied to the column.Elution is with Eluent A at a flow rate of 0.5 ml/min for 30 min, anddetection is achieved by measuring absorbance at 215 nm. The content ofaggregates is calculated as the peak area of the Factor VIIaggregates/total area of Factor VII peaks (monomer and aggregates).

“Bioavailability” refers to the proportion of an administered dose of aFactor VII or Factor VII-related preparation that can be detected inplasma at predetermined times after administration. Typically,bioavailability is measured in test animals by administering a dose ofbetween about 25-250 μg/kg of the preparation; obtaining plasma samplesat predetermined times after administration; and determining the contentof Factor VII or Factor VII-related polypeptides in the samples usingone or more of a clotting assay (or any bioassay), an immunoassay, or anequivalent. The data are typically displayed graphically as [Factor VII]v. time and the bioavailability is expressed as the area under the curve(AUC). Relative bioavailability of a test preparation refers to theratio between the AUC of the test preparation and that of the referencepreparation.

In some embodiments, the preparations of the present invention exhibit arelative bioavailability of at least about 110%, preferably at leastabout 120%, more preferably at least about 130% and most preferably atleast about 140% of the bioavailability of a reference preparation. Thebioavailability may be measured in any mammalian species, preferablydogs, and the predetermined times used for calculating AUC may encompassdifferent increments from 10 min-8 h.

“Half-life” refers to the time required for the plasma concentration ofFactor VII polypeptides of Factor VII-related polypeptides to decreasefrom a particular value to half of that value. Half-life may bedetermined using the same procedure as for bioavailability. In someembodiments, the preparations of the present invention exhibit anincrease in half-life of at least about 0.25 h, preferably at leastabout 0.5 h, more preferably at least about 1 h, and most preferably atleast about 2 h, relative to the half-life of a reference preparation.

“Immunogenicity” of a preparation refers to the ability of thepreparation, when administered to a human, to elicit a deleteriousimmune response, whether humoral, cellular, or both. Factor VIlapolypeptides and Factor VIIa-related polypeptides are not known toelicit detectable immune responses in humans. Nonetheless, in any humansub-population, there may exist individuals who exhibit sensitivity toparticular administered proteins. Immunogenicity may be measured byquantifying the presence of anti-Factor VII antibodies and/or FactorVII-responsive T-cells in a sensitive individual, using conventionalmethods known in the art. In some embodiments, the preparations of thepresent invention exhibit a decrease in immunogenicity in a sensitiveindividual of at least about 10%, preferably at least about 25%, morepreferably at least about 40% and most preferably at least about 50%,relative to the immunogenicity for that individual of a referencepreparation.

Pharmaceutical Compositions and Methods of Use The preparations of thepresent invention may be used to treat any Factor VII-responsivesyndrome, such as, e.g., bleeding disorders, including, withoutlimitation, those caused by clotting factor deficiencies (e.g.,haemophilia A and B or deficiency of coagulation factors XI or VII); bythrombocytopenia or von Willebrand's disease, or by clotting factorinhibitors, or excessive bleeding from any cause. The preparations mayalso be administered to patients in association with surgery or othertrauma or to patients receiving anticoagulant therapy.

Preparations comprising Factor VII-related polypeptides according to theinvention, which have substantially reduced bioactivity relative towild-type Factor VII, may be used as anticoagulants, such as, e.g., inpatients undergoing angioplasty or other surgical procedures that mayincrease the risk of thrombosis or occlusion of blood vessels as occurs,e.g., in restenosis. Other medical indications for which anticoagulantsare prescribed include, without limitation, deep vein thrombosis,pulmonary embolism, stroke, disseminated intravascular coagulation(DIC), fibrin deposition in lungs and kidneys associated withgram-negative endotoxemia, myocardial infarction; Acute RespiratoryDistress Syndrome (ARDS), Systemic Inflammatory Response Syndrome(SIRS), Hemolytic Uremic Syndrome (HUS), MOF, and TTP.

Pharmaceutical compositions comprising the Factor VII and FactorVII-related preparations according to the present are primarily intendedfor parenteral administration for prophylactic and/or therapeutictreatment. Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly.They may be administered by continuous or pulsatile infusion.

Pharmaceutical compositions or formulations comprise a preparationaccording to the invention in combination with, preferably dissolved in,a pharmaceutically acceptable carrier, preferably an aqueous carrier ordiluent. A variety of aqueous carriers may be used, such as water,buffered water, 0.4% saline, 0.3% glycine and the like. The preparationsof the invention can also be formulated into liposome preparations fordelivery or targeting to the sites of injury. Liposome preparations aregenerally described in, e.g., U.S. Pat. Nos. 4,837,028, 4,501,728, and4,975,282. The compositions may be sterilised by conventional,well-known sterilisation techniques. The resulting aqueous solutions maybe packaged for use or filtered under aseptic conditions andlyophilised, the lyophilised preparation being combined with a sterileaqueous solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances or adjuvants, including, without limitation, pH adjusting andbuffering agents and/or tonicity adjusting agents, such as, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, etc.

The concentration of Factor VII or Factor VII-related polypeptides inthese formulations can vary widely, i.e., from less than about 0.5% byweight, usually at or at least about 1% by weight to as much as 15 or20% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution and 10mg of the preparation. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.(1990).

The compositions containing the preparations of the present inventioncan be administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a subjectalready suffering from a disease, as described above, in an amountsufficient to cure, alleviate or partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as“therapeutically effective amount”. Effective amounts for each purposewill depend on the severity of the disease or injury as well as theweight and general state of the subject. In general, however, theeffective amount will range from about 0.05 mg up to about 500 mg of thepreparation per day for a 70 kg subject, with dosages of from about 1.0mg to about 200 mg of the preparation per day being more commonly used.It will be understood that determining an appropriate dosage may beachieved using routine experimentation, by constructing a matrix ofvalues and testing different points in the matrix.

Local delivery of the preparations of the present invention, such as,for example, topical application, may be carried out, e.g., by means ofa spray, perfusion, double balloon catheters, stent, incorporated intovascular grafts or stents, hydrogels used to coat balloon catheters, orother well established methods. In any event, the pharmaceuticalcompositions should provide a quantity of the preparation sufficient toeffectively treat the subject.

The pharmaceutical compositions of the invention may further compriseother bioactive agents, such as, e.g., non-Factor VII-related coagulantsor anticoagulants.

The following examples are intended as non-limiting illustrations of thepresent invention.

EXAMPLE1 Production and Analysis of a Factor VII Preparation Exhibitingan Altered Glycoform Pattern

The following experiment was performed to produce a Factor VIIpreparation having an altered glycoform pattern.

I. Production: A BHK cell line transformed with a Factor VII-encodingplasmid was adapted to growth in suspension culture in the absence ofserum. The cells were propagated sequentially in spinner cultures and asthe cell number increased, the volume was gradually increased byaddition of new medium.

Finally, 6 l of seed culture were inoculated into a 100-liter productionbioreactor containing macroporous Cytopore 1 carriers (Pharmacia), afterwhich the suspension cells became immobilized in the carriers. Theculture was maintained at 36° C. at a pH of 6.7-6.9 and a DO of 50%. Thevolume in the production bioreactor was gradually increased by additionof new medium as the cell number increased. When the cell densityreached approximately 2×10⁶ cells/ml, the production phase was initiatedand a medium change was performed every 24 hours: Agitation was stoppedto allow for sedimentation of the cell-containing carriers, and 80% ofthe culture supernatant was then harvested and replaced with new medium.The harvested culture supernatant was filtered to remove non-trappedcells and cell debris and was then transferred for further processing.

During the production phase the cells reached 3-6×10⁶ cells/ml and atiter of 2-7 mg Factor VII/liter.

II. Analysis of the Glycoform Pattern of Recombinant Factor VII

The oligosaccharide patterns of the following preparations werecompared: (a) recombinant Factor VII preparations produced as describedin part I (n=7); and two reference preparations: (b) recombinant FactorVII preparations produced in BHK cells in the presence of calf serum(n=10); and (c) a Factor VII preparation purified from human plasma.

The N-linked oligosaccharides were released from the polypeptides bychemical cleavage (hydrazinolysis, on a GlycoPrep1000 unit, OxfordGlycoSciences) or by enzymatic cleavage (N-glycosidase F from, eg.,Boehringer Mannheim). The released oligosaccharides were labeled with2-aminobenzamide (using a signal labelling kit, K-404, OxfordGlycoSciences or Glyko). The labeled oligosaccharides were analysedusing anion-exchange HPLC on a CarboPac PA100 column (4×250 mm, Dionex,P/N 43055) with a Guard column (4×50 mm, Dionex, P/N 43054). The columnwas equilibrated with 150 mM sodium hydroxide and eluted with a gradientof 0-150 mM sodium acetate, 150 mM sodium hydroxide. Oligosaccharideswere detected using fluorescence, with excitation at 330 nm and emissionat 420 nm.

The relative contents of the various Factor VII oligosaccharidestructures (Klausen et al., 1998) were calculated as the relative peakareas for the carbohydrate peaks in the anion-exchange HPLC analysis.Based on the structural elements of each oligosaccharide, it wasassigned to one of the following: (i) chains containing at least onesialic acid; (ii) chains lacking any sialic acid (i.e., neutral); (iii)chains containing at least one terminal galactose residue; (iv) chainscontaining at least one terminal N-acetylgalactosamine residue; and (v)chains containing at least one uncapped antenna (i.e., at least oneterminal galactose or N-acetylgalactosamine residue). Finally, the sumof the relative contents of the oligosaccharide chains assigned to eachgroup was calculated as a percentage of the total oligosaccharidechains. The standard deviation of this determination was calculated tobe 0.08% (intraday variation); 0.7% (day-to-day variation); and 0.5%(1-100 μg interval).

The resulting glycoform patterns are illustrated in the following table:(i) (ii) (iii) (iv) (v) a 93.1-98.7 1.3-6.9  5.9-16.4 5.9-8.7 11.7-23.9b 88.3-92.5  7.5-12.9  9.4-16.8 19.0-28.6 30.1-39.0 c 99.5% <0.5%   2-3% 0%    2-3%

The recombinant Factor VII preparations produced according to thisExample (i.e., in the absence of serum) exhibit a glycoform pattern thatdiffers from both the glycoform pattern of recombinant Factor VIIproduced in the presence of serum and native Factor VII isolated fromhuman plasma. The oligosaccharides of recombinant Factor VII produced inthe absence of serum are sialylated to a higher extent than thoseproduced in the presence of serum and contain less neutral chains andless chains that terminate in either galactose or N-acetylgalatosamine.

III. Bioavailability:

The following experiment was performed to compare the bioavailability oftwo Factor VII preparations produced as above (I and II) with that oftwo reference Factor VII preparations (i.e., produced in the presence ofserum) (A and B).

Groups of 8 rats were administered either a test preparation or areference preparation at a dose of 25 μg/kg (≈100 μg/rat) in aglycylglycine buffer (pH 7.4) containing sodium chloride (7.87 mg/ml),calcium chloride dihydrate (1.48 mg/ml), mannitol (2.5 mg/ml) andpolysorbate 80. Blood samples were withdrawn at 10 min and 30 minfollowing the initial administration. Plasma was obtained from thesamples and Factor VII was quantified by ELISA. Bioavailability of eachsample is expressed as the dose-adjusted area under the plasmaconcentration curve for Factor VII based on the 10 and 30-min samples(AUC₁₀₋₃₀/dose). The relative bioavailability is expressed as the ratiobetween the mean AUC₁₀₋₃₀/dose of the test and reference samples × 100.The 90% confidence limits for the relative bioavailability werecalculated from the 90% confidence limits for differences betweenpreparations.

The results are summarized in the Table below. (The % sialylation ofeach preparation, which was measured as described above, is indicated inparentheses). relative 90% conf. 90% conf. test referencebioavailability lower upper I A 128.6 116.1 141.1 (97.5%) (93%) I B154.9 141.2 168.5 (97.5%) (86%) II A 117.3 104.8 129.8 (96.7%) 93% II B141.2 127.5 154.8 (96.7%) (86%)

The results indicate that even relatively small differences in theproportion of oligosaccharide chains having at least one sialic acidresidue, such as, e.g., between 93% and 96 or 97%, can have a markedimpact on bioavailability (increase of 20-30%). A 10% increase in the %sialylation, moreover, causes a 40-50% increase in bioavailability.

EXAMPLE2 Analysis of Factor VII Preparations Exhibiting an AlteredGlycoform Pattern

Factor VII was produced as described in Example 1 above, with theexception that the Factor VII was harvested from 500-1 cultures.Glycoform analysis was performed as described in Example 1. Threeindependent preparations (A, B, and C) were analyzed and compared with areference preparation (D).

Bioavailability was measured in a dog model as follows: The experimentwas performed as a four leg cross-over study in 12 Beagle dogs dividedin four groups. All animals received each of the three test preparationsA, B, and C and the reference preparation D at a dose of ≈90 μg/kg in aglycylglycine buffer (pH 5.5) containing sodium chloride (2.92 mg/ml),calcium chloride dihydrate (1.47 mg/ml), mannitol (30 mg/ml) andpolysorbate 80. Blood samples were withdrawn at 10, 30, and 60 minutesand 2, 3, 4, 6 and 8 hours following the initial administration. Plasmawas obtained from the samples and Factor VII was quantified by ELISA.

Bioavailability of each sample is expressed as the dose-adjusted areaunder the plasma concentration curve for Factor VII (AUC/dose). Therelative bioavailability is expressed as the ratio between the meanAUC/dose of the test and reference preparation ×100 and 90% confidencelimits for the relative bioavailability were calculated.

The results are summarized in the Table below. The % sialylation of eachpreparation, which was measured as described in Example 1 above, isindicated in parentheses. Relative 90% conf. limit 90% conf. limit TestReference bioavailability lower upper A D 144 135 153 (98.7%) (88.2%) BD 127 119 136 (95.9%) (88.2%) C D 112 105 120 (93.1%) (88.2%)

The results indicate that small differences in the proportion ofoligosaccharide chains having at least one sialic acid residue have amarked impact on bioavailability of Factor VII. A 10% increase in the %sialylation causes a 30-50% increase in bioavailability with a close tolinear relationship for the three test preparations and the referencepreparation

EXAMPLE 3 Factor VII Preparations Exhibiting an Altered GlycoformPattern

The following experiment was performed to produce a Factor VIIpreparation having an altered glycoform pattern.

I. Construction of Cell Line and Factor VII Production:

A plasmid vector pLN174 for expression of human FVII has been described(Persson and Nielsen. 1996. FEBS Lett. 385: 241-243). Briefly, itcarries the cDNA nucleotide sequence encoding human FVII including thepropeptide under the control of a mouse metallothionein promoter fortranscription of the inserted cDNA, and mouse dihydrofolate reductasecDNA under the control of an SV40 early promoter for use as a selectablemarker.

For construction of a plasmid vector encoding a gamma-carboxylationrecognition sequence, a cloning vector (pBluescript II KS+, Stratagene)containing cDNA encoding FVII including its propeptide was used(pLN171). (Persson et al. 1997. J. Biol. Chem. 272: 19919-19924). Anucleotide sequence encoding a stop codon was inserted into the cDNAencoding FVII after the propeptide of FVII by inverse PCR-mediatedmutagenesis using this cloning vector. The template plasmid wasdenatured by treatment with NaOH followed by PCR with Pwo(Boehringer-Mannheim) and Taq (Perkin-Elmer) polymerases with thefollowing primers: a) 5′-AGC GTT TTA GCG CCG GCG CCG (SEQ ID NO. 19) GTGCAG GAC-3′ b) 5′-CGC CGG CGC TAA AAC GCT TTC (SEQ ID NO. 20) CTG GAG GAGCTG CGG CC-3′

The resulting mix was digested with DpnI to digest residual template DNAand Escherichia coli were transformed with the PCR product. Clones werescreened for the presence of the mutation by sequencing. The cDNA from acorrect clone was transferred as a BamHI-EcoRI fragment to theexpression plasmid pcDNA3 (Invitrogen). The resulting plasmid was termedpLN329. CHO K1 cells (ATCC CCl61) were transfected with equal amounts ofpLN174 and pLN329 with the Fugene6 method (Boehriner-Mannheim).Transfectants were selected by the addition of methotrexate to 1 μM andG-418 to 0.45 mg/ml. The pool of transfectants were cloned by limitingdilution and FVII expression from the clones was measured.

A high producing clone was further subcloned and a clone E11 with aspecific FVII expression of 2.4 pg/cell/day in Dulbecco-modified Eagle'smedium with 10% fetal calf serum was selected. The clone was adapted toserum free suspension culture in a commercially available CHO medium(JRH Bioscience) free of animal derived components.

The adapted cells were propagated sequentially in spinner cultures andas the cell number increased, the volume was gradually increased byaddition of new medium. After 25 days, 6 1 of spinner culture wereinoculated into a 50-liter bioreactor. The cells were propagated in thebioreactor and as the cell number increased, the volume was graduallyincreased by addition of new medium.

Finally, 50 1 of seed culture were inoculated into a 500-literproduction bioreactor containing macroporous Cytopore 1 carriers(Pharmacia), after which the suspension cells became immobilized in thecarriers. The culture was maintained at 36□ C. at a pH of 7.0-7.1 and aDissolved Oxygen Tension (DOT) of 50% of saturation. The volume in thebioreactor was gradually increased by addition of new medium as the cellnumber increased. When the cell density reached approximately 10-12×10⁵cells/ml, the production phase was initiated and a medium change wasperformed every 24 hours: agitation was stopped to allow forsedimentation of the cell-containing carriers, and 80% of the culturesupernatant was then harvested and replaced with new medium. Theharvested culture supernatant was filtered to remove non-trapped cells(i.e. cells that were not immobilized in carriers) and cell debris andwas then transferred for further processing.

During the production phase the cells reached 2-3×10⁷ cells/ml and atiter of 8 mg Factor VII/liter.

II. Glycoform Analysis:

A. The oligosaccharide pattern of a Factor VII preparation produced asdescribed above (a) was compared with those of (b) recombinant FactorVII preparations produced in BHK cells in the presence of calf serum and(c) a Factor VII preparation purified from human plasma. The methodsused were essentially as described in Example 1.

The results are shown in the Table below. The oligosaccharideassignments are as follows: (i) chains containing at least one sialicacid; (ii) chains lacking any sialic acid (i.e., neutral); (iii) chainscontaining at least one terminal galactose residue; (iv) chainscontaining at least one terminal N-acetylgalactosamine residue; and (v)chains containing at least one uncapped antenna (i.e., at least oneterminal galactose or N-acetylgalactosamine residue). (i) (ii) (iii)(iv) (v) A 95.2 4.8 22.9 0.1 23.0 B 88.3-92.5 7.5-12.9  9.4-16.819.0-28.6 30.1-39.0 C 99.5% <0.5%    2-3% 0%    2-3%

B. The oligosaccharide patterns of five independent Factor VIIpreparations produced as described in this Example (a) were comparedwith those of (b) recombinant Factor VII preparations produced in BHKcells in the presence of calf serum and (c) a Factor VII preparationpurified from human plasma, using the analytical methods described inExample 1.

Based on the structural elements of each oligosaccharide, it wasassigned to one of the following: (i) chains containing at least onesialic acid; (ii) chains lacking any sialic acid (i.e., neutral); (iii)chains containing at least one fucose linked to the antenna. Finally,the sum of the relative contents of the oligosaccharide chains assignedto each group was calculated as a percentage of the totaloligosaccharide chains. The standard deviation of this determination wascalculated to be 0.08% (intraday variation); 0.7% (day-to-dayvariation); and 0.5% (1-100 μg interval).

The resulting glycoform patterns are illustrated in the following Table:(i) (ii) (iii) A 89.0-97.9% 2.1-11.0% 6.3-21.3% B 88.3-92.5% 7.5-12.9%0% C 99.5% <0.5% 0%

The recombinant Factor VII preparations produced according to Example 1(i.e., produced in the absence of serum by the CHO cell line) exhibit aglycoform pattern that differs from both the glycoform pattern ofrecombinant Factor VII produced in the presence of serum and nativeFactor VII isolated from human plasma. The oligosaccharides ofrecombinant Factor VII produced in the absence of serum by the CHO 282.4cell line include structures with fucose linked to the antenna, whichare absent from both of the reference preparations. Two of thestructures have been purified and characterized by matrix assisted laserdesorption ionisation mass spectrometry, by treatment with linkagespecific fucosidase enzymes and by anion-exchange HPLC as describedabove. The two structures have been shown to contain the sialyl Lewis xstructure, i.e., fucose linked α1->3 to an antennary N-acetylglucosaminein a sialylated oligosaccharide.

III. Bioactivity:

Five Factor VII preparations produced as described in this Example wereanalyzed for (a) thrombin generation and (b) binding to tissue factor(TF) and compared with recombinant Factor VII produced in BHK cells inthe presence of serum (reference). The following Table correlates theglycoform patterns (% of oligosaccharide chains containing sialic acidand the % containing fucosylated antenna) and the two bioactivities.Thrombin Oligosaccharide generation Factor VII Pattern (% of TF bindingPreparation % Sialyl % Fucosyl reference) Kd (nM) 1 98  6 125 2.8 2 9413 123 2.0 3 93 14 126 1.8 4 88 16 145 3.3 5 86 21 158 2.8 reference86-93  0 100 2.2-6.6

The results indicate that Factor VII preparations having fucosylatedantennae exhibit higher TF-independent Factor VII activity (asexhibited, e.g. by thrombin generation) than Factor VII preparationslacking fucosylated antennae.

EXAMPLE 4 In Vitro Hydrolysis Assay

The following method can be used to assay Factor VIIa bioactivity. Theassay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).The chromogenic substrate D-Ile-Pro-Arg-p-nitroanilide (S-2288,Chromogenix, Sweden), at a final concentration of 1 mM, is added toFactor VIIa (final concentration 100 nM) in 50 mM Hepes, pH 7.4,containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin. Theabsorbance at 405 nm is measured continuously in a SpectraMax™ 340 platereader (Molecular Devices, USA). The absorbance developed during a20-minute incubation, after subtraction of the absorbance in a blankwell containing no enzyme, is used to calculate the ratio between theactivities of a test and a reference Factor VIIa.

EXAMPLE 5 In Vitro Proteolysis Assay

The following method can be used to assay Factor VIIa bioactivity. Theassay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark).Factor VIIa (10 nM) and Factor ×(0.8 microM) in 100 μl 50 mM Hepes, pH7.4, containing 0.1 M NaCl, 5 mM CaCl₂ and 1 mg/ml bovine serum albumin,are incubated for 15 min. Factor X cleavage is then stopped by theaddition of 50 μl 50 mM Hepes, pH 7.4, containing 0.1 M NaCl, 20 mM EDTAand 1 mg/ml bovine serum albumin. The amount of Factor Xa generated ismeasured by addition of the chromogenic substrateZ-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), finalconcentration 0.5 mM. The absorbance at 405 nm is measured continuouslyin a SpectraMax™ 340 plate reader (Molecular Devices, USA). Theabsorbance developed during 10 minutes, after subtraction of theabsorbance in a blank well containing no FVIIa, is used to calculate theratio between the proteolytic activities of a test and a referenceFactor VIIa.

All patents, patent applications, and literature references referred toherein are hereby incorporated by reference in their entirety.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed description.Such obvious variations are within the full intended scope of theappended claims.

1. A method for large-scale production of Factor VII or a FactorVII-related polypeptide, comprising the steps of: (i) propagating alarge-scale culture of mammalian cells in medium lacking animal-derivedcomponents until the large-scale culture cells reach a secondpredetermined density, said large-scale culture having been created by amethod comprising: inoculating mammalian cells expressing Factor VII ora Factor VII-related polypeptide into a seed culture vessel containingmedium lacking animal-derived components; propagating the inoculatedcells at least until the cells have reached a first predetermineddensity to form a seed culture, transferring the seed culture to alarge-scale culture vessel containing medium lacking animal derivedcomponents to form said large-scale culture; (ii) maintaining thelarge-scale culture in medium lacking animal-derived components underconditions appropriate for Factor VII expression, thereby causing thecells to produce Factor VII or a Factor VII-related polypeptide, and(iii) recovering the produced Factor VII or Factor VII-relatedpolypeptide from the maintained culture.
 2. A method as defined in claim1, wherein said cells are CHO cells.
 3. A method as defined in claim 1,wherein said Factor VII has a glycosylation pattern different from bothFactor VII produced in vivo and Factor VII produced in BHK cells.
 4. Amethod as defined in claim 1, wherein said seed culture has beentransferred to and propagated in one or more intermediate size vesselsof progressively larger size prior to being transferred to said largescale vessel.
 5. A method as defined in claim 1, wherein the cells havebeen rendered suspension culture competent prior to being inoculatedinto the seed vessel.
 6. A method as defined in claim 1, wherein thecells have been adapted to grow in medium lacking animal-derivedcomponents prior to said inoculation.
 7. A method as defined in claim 1,wherein the large-scale culture is a macrocarrier culture.
 8. A methodas defined in claim 7, wherein the large-scale culture is a macroporouscarrier culture, said macroporous carrier bearing a positive charge. 9.A method as defined in claim 1, wherein the maintaining step comprisesregularly harvesting a portion of the supernatant of said large-scaleculture and replacing it with fresh medium lacking animal-derivedcomponents.
 10. A method as defined in claim 1, wherein the maintainingstep comprises sedimentation of the cell-containing carriers prior tosaid harvesting.
 11. A method as defined in claim 1, wherein themaintaining step comprises cooling the culture to a pre-determinedtemperature before the sedimentation.
 12. A method as defined in claim1, wherein the maintaining step comprises feeding said cells withglucose.
 13. A method as defined in claim 12, wherein feeding comprisespulse feeding from 1 to 4 times per 24-hour period.
 14. A method asdefined in claim 12, wherein said feeding comprises gradual orcontinuous introduction of glucose into the large scale culture.
 15. Amethod for large-scale production of Factor VII or a Factor VII-relatedpolypeptide, comprising the steps of: (i) maintaining a large-scaleculture of mammalian cells having a second predetermined density inmedium lacking animal-derived components under conditions appropriatefor Factor VII expression, thereby causing the cells to produce FactorVII or a Factor VII-related polypeptide, said large-scale culture havingbeen created by a method comprising: inoculating mammalian cellsexpressing Factor VII or a Factor VII-related polypeptide into a seedculture vessel containing medium lacking animal-derived components;propagating the inoculated cells at least until the cells have reached afirst predetermined density to form a seed culture, transferring theseed culture to a large-scale culture vessel containing medium lackinganimal derived components to form said large-scale culture; and (ii)recovering produced Factor VII or Factor VII-related polypeptide fromthe maintained culture.
 16. A method for large-scale production ofFactor VII or a Factor VII-related polypeptide, comprising the steps of:(i) maintaining a large-scale culture of mammalian cells having a secondpredetermined density in medium lacking animal-derived components underconditions appropriate for Factor VII expression, thereby causing thecells to produce Factor VII or a Factor VII-related polypeptide, saidlarge-scale culture having been created by a method comprising:inoculating mammalian cells expressing Factor VII or a FactorVII-related polypeptide into a seed culture vessel containing mediumlacking animal-derived components; propagating the inoculated cells atleast until the cells have reached a first predetermined density to forma seed culture, and (ii) transferring the seed culture to a large-scaleculture vessel containing medium lacking animal derived components toform said large-scale culture.
 17. A Factor VII or Factor VII-relatedpolypeptide produced by a method as defined in claim
 1. 18. A Factor VIIor Factor VII-related polypeptide produced by a method as defined inclaim
 15. 19. A Factor VII or Factor VII-related polypeptide produced bya method as defined in claim
 16. 20. A preparation comprising aplurality of Factor VII or Factor VII-related polypeptides expressed byrecombinant BHK or CHO cells in the presence of media lackinganimal-derived components (serum-free Factor VII), wherein the FactorVII or Factor VII-related polypeptides comprise N-linkedoligosaccharides chains and the oligosaccharides exhibit a glycoformpattern differing from that of the same Factor VII or Factor VII-relatedpolypeptide expressed by the same cells in the presence of serum(serum-raised Factor VII) and from that of Factor VII purified fromhuman plasma (native Factor VII) and wherein a percentage ofoligosaccharide chains in said preparation comprise at least one sialicacid moiety, said percentage being higher than that observed inserum-raised Factor VII preparations and lower than the correspondingpercentage in native Factor VII preparations, said serum-free Factor VIIpreparation having a higher bioavailability than the bioavailability ofa serum-raised Factor VII preparation.
 21. A preparation as defined inclaim 20, wherein said serum-free Factor VII glycoform pattern exhibitsan additional difference from that of Factor VII expressed by the samecells in the presence of serum (serum-raised Factor VII) and from thatof Factor VII purified from human plasma (native Factor VII), saidadditional difference comprising one or more of the following: (i)percentage of the oligosaccharide chains having a neutral charge,wherein the percentage of oligosaccharide chains of serum-free FactorVII having a neutral charge is lower than that of serum-raised FactorVII and higher than that of native Factor VII; (ii) percentage of theoligosaccharide chains comprising at least one terminal galactoseresidue, wherein the percentage of oligosaccharide chains of serum-freeFactor VII having a at least one terminal galactose residue is lowerthan that of serum-raised Factor VII and higher than that of nativeFactor VII; (iii) percentage of the oligosaccharide chains comprising atleast one terminal N-acetylgalactosamine residue, wherein the percentageof oligosaccharide chains of serum-free Factor VII having a at least oneterminal N-acetyl galactose residue is lower than that of serum-raisedFactor VII and higher than that of native Factor VII; and (iv)percentage of the oligosaccharide chains comprising at least oneuncapped antenna, wherein the percentage of oligosaccharide chains ofserum-free Factor VII comprising at least one uncapped antenna is lowerthan that of serum-raised Factor VII and higher than that of nativeFactor VII.
 22. A pharmaceutical formulation comprising a polypeptide asdefined in claim 17 and a pharmaceutically acceptable carrier oradjuvant.
 23. A method for treating a Factor VII-responsive syndrome,the method comprising administering a pharmaceutical formulation asdefined in claim 22 to a patient in need of such treatment, underconditions that result in a decrease in bleeding and/or an increase inblood clotting.
 24. A method as defined in claim 23, wherein thesyndrome is selected from the group consisting of haemophilia A,haemophilia B, Factor XI deficiency, Factor VII deficiency,thrombocytopenia, von Willebrand's disease, presence of a clottingfactor inhibitor, surgery, trauma, and anticoagulant therapy.